The present invention relates to a mutated trypsin comprising an amino acid substitution both at position K60 and D189, and at least one more amino acid substitution by histidine at position N143 or position E151. Such trypsin mutant has a preferred cleavage site comprising the amino acids Xaa1-Xaa2-His, wherein Xaa1 is L, Y or F and Xaa2 is R or K. The invention also relates to a man-made polypeptide comprising a target peptide and the above cleavage site as well as to a method of producing C-terminally modified target peptides by using this mutated trypsin.
The use of biologically active peptides, e.g. for pharmaceutical purposes has become more and more important during the past years. Several methods exist to produce such biologically active peptides, for example, the chemical synthesis based on solid phase or solution phase peptide synthesis techniques, or the cultivation of genetically manipulated microorganisms followed by the isolation and purification of such produced recombinant proteins.
However, it remains difficult and costly to chemically synthesize polypeptides of more than about 50 amino acids. It also represents a significant task to modify a peptide obtained by chemical peptide synthesis and/or a recombinantly obtained polypeptide at its C-terminal end. One of the most powerful methods to modify polypeptides is through a controlled protein ligation, whereby peptide analogs, unnatural amino acids, stable isotopes, fluorophores, and other biochemically or biophysically important molecules can be specifically incorporated into a polypeptide. One of these methods is based on the introduction—mostly synthetically—of a chemo-selective amino acid, mainly a cysteine which then is modified by a thio-selective reagent attacking the SH-side chain of this amino acid residue. A further alternative is the so-called intein-based protein ligation system, which can generate a protein thioester by proteolysis of a corresponding protein-intein fusion protein (Blaschke, U.K., et al., Methods Enzymol. 328 (2000) 478-496). This method has been successfully applied to introduce unnatural modifications into proteins. However, difficulties remain, e.g., because the target protein must be expressed as a fusion protein together with an intein.
Recently a few more enzyme-based approaches for peptide ligation and/or C-terminal modification have been described. Breddam and co-workers (e.g., U.S. Pat. No. 5,985,627) describe the use of the serine protease carboxypeptidase Y (CPD-Y) in the C-terminal modification of peptides with fluorescence or affinity labels. This modification is based on the specific ability of CPD-Y to stepwise cleave amino acids off the C-terminal end of polypeptides. This method therefore may be considered to be a specific tool for modification of the C-terminus of a polypeptide. CPD-Y cleaves off the C-terminal amino acid under formation of a peptide-acyl-enzyme-intermediate. This acyl-enzyme-intermediate upon nucleophilic attack is deacylated resulting in a transamidation reaction. The desired transamidation reaction may be accompanied by (un-wanted) side reactions like hydrolysis. It is also possible that more than one C-terminal amino acid is cleaved off, on the other hand also amino acids may be added by this method (Stennicke, H. R., et al., Anal. Biochem. 248 (1997) 141-148 and Buchardt, O., et al., U.S. Pat. No. 5,580,751)
Abrahmson et al. (e.g. WO 94/18329) described the use of serine protease variants for ligation of peptides. Subtilisin variants are disclosed which have an improved peptide ligase activity. It is, however, necessary for effective peptide ligation to use an appropriate amino terminus protecting group and an appropriate carboxy terminus activating group, respectively, in order to efficiently ligate two peptide substrates.
Recently sortase-mediated protein ligation has been described as an alternative method in protein engineering (Mao, H., et al., J. Am. Chem. Soc. 126 (2004) 2670-2671). Sortase, an enzyme isolated from Staphylococcus aureus catalyses a transpeptidation reaction by cleaving between threonine and glycine in a recognition motif consisting of the amino acids LPXTG (SEQ ID NO: 20) and subsequently joining the carboxyl group of threonine to an N-terminal glycine. In nature it catalyses the transpeptidation of the threonine to an amino group of pentaglycine on the cell wall peptidoglycan.
In the sortase recognition motif LPXTG (SEQ ID NO: 20), X may be the amino acids D, E, A, N, Q, or K. This enzyme has been used to ligate carboxy terminal threonine residues of a peptide or a protein to an N-terminal glycine of a second peptide. As mentioned, sortase requires a recognition motif of five amino acids of which four amino acids (LPXT) will be present within the ligation product.
Therefore, whereas several methods exist for C-terminal modification of polypeptides there is a tremendous need for alternative or improved methods of C-terminal modification of polypeptides. The inventors of the present invention have found that it is possible to use special trypsin mutants in the C-terminal modification of polypeptides.